Human enbryonic germ cell and methods of use

ABSTRACT

Primordial germ cells extracted from post blastocyst human embryos, such as from the gonadal ridges of a 8-11 week LMP human embryo, are disclosed. The primordial germ cells are cultured in long term culture (more than 30 days) resulting in cells that resemble embryonic stem cells in morphology and pluripotency. The cells are maintained several months in culture and can be genetically manipulated using transgenic technology to insert heterologous genetic material.

This is a continuation-in-part of U.S. application Ser. No. 08/829,372,filed Mar. 31, 1997 (pending) the disclosure of which is incorporatedherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of in vitro culture ofundifferentiated cells and methods of producing such cells. Morespecifically, the invention relates to methods and compositions forproduction of human pluripotent embryonic germ cells or (hEG) celllines.

2. Description of Related Art

Pluripotent embryonic stem cells are derived principally from twoembryonic sources. In the mouse, one type of pluripotent stem cell canbe isolated from cells of the inner cell mass of a pre-implantationembryo and are termed embryonic stem (ES) cells (Evans and Kaufman,Nature 292: 154-156, 1981). A second type of mouse pluripotent stem cellcan be isolated from primordial germ cells (PGCs) located in the genitalridges of day 8.5 post coitum mouse embryos and has been termed anembryonic germ cell (EG) (Matsui et al., Nature 353: 750-751, 1991;Resnick et al., Nature 359: 550-551, 1992; Hogan, U.S. Pat. No.5,453,357). Both types of cells are pluripotent and demonstrate germlinegenetic transmission in the mouse.

The extent of pluripotency in pluripotent cell cultures is generallydetermined experimentally. For example, one method utilizes measuringthe high intracellular levels of the enzyme alkaline phosphatase foundin ES, PGCs, and EGs. Demonstration of intracellular alkalinephosphatase by histological staining was historically used to define andlocate PGCs (Chiquoine, Anat.Rec. 118: 135-146, 1954). Such stainingremains one of the criteria for the definition of new pluripotent cellcultures.

ES and EGs propagated in vitro can contribute efficiently to theformation of chimeras, including germline chimeras, but in addition,both of these cell types can be genetically manipulated in vitro withoutlosing their capacity to generate germ-line chimeras.

ES and EGs are useful in methods for the generation of transgenicanimals. Such methods have a number of advantages as compared with moreconventional techniques for introducing new genetic material into suchanimals, such as zygote injection and viral infection. First, the geneof interest can be introduced and its integration and expressioncharacterized in vitro. Second, the effect of the introduced gene on theES or EG growth can be studied in vitro. Third, the characterized ES orEGs having a novel introduced gene can be efficiently introduced intoembryos by blastocyst injection or embryo aggregation and theconsequences of the introduced gene on the development of the resultingtransgenic chimeras monitored during prenatal or postnatal life. Fourth,the site in the ES or EG genome at which the introduced gene integratescan be specified, permitting subsequent gene targeting and genereplacement (Thomas, K. R. and Capecci, M. R. Cell 51: 503-512, 1987).

However, it is known that EGs or ES cells and certain EC (embryonalcarcinoma) cell lines will only retain the stem cell phenotype in vitrowhen cultured on a feeder layer of fibroblasts (such as murine STOcells, e.g., Martin, G. R. and Evans, M. J. Proc. Natl. Acad. Sci. USA72: 1441-1445, 1975) when cultured in medium conditioned by certaincells (e.g. Koopman, P. And Cotton, R. G. H. Exp. Cell 154: 233-242,1984; Smith, A. G. and Hooper, M. L. Devel. Biol. 121: 1-91, 1987) or bythe exogenous addition of leukemia inhibitory factor (LIF). Such cellscan be grown relatively indefinitely using the appropriate cultureconditions. They can be induced to differentiate in vitro using retinoicacid or spontaneously by removal of the feeder layer conditioned mediaor exogenous LIF. In addition, these cells can be injected into a mouseblastocyst to form a somatic and germ line chimera. This latter propertyhas allowed mouse ES cells to be used for the production of transgenicmice with specific changes to the genome.

See M. Evans et al., Nature 292: 154 (1981); G. Martin, Proc. Natl. AcadSci. USA 78: 7638 (1981); A. Smith et al., Developmental Biology 121: 1(1987); T. Doetschman et al., Developmental Biology 127: 224 (1988); A.Handyside et al., Roux's Arch Dev. Biol. 198: 48 (1989).

In the absence of feeder cells or conditioned medium, ES or EGsspontaneously differentiate into a wide variety of cell types,resembling those found during embryogenesis and in the adult animal.With the appropriate combinations of growth and differentiation factors,mouse ES and EGs generate cells of the hematopoietic lineage in vitro(Keller, G., et al., Mol. Cell. Biol. 13: 473-486, 1993; Palacios, R.,E. Golunski, and J. Samaridis, Proc. Natl. Acad. Sci. USA 92: 7530-7534,1995; Rich, T., Blood 86: 463-472, 1995). Additionally, mouse ES cellshave been used to generate in vitro cultures of neurons (Bain, G., etal., Developmental Biology 168: 342-357, 1995; Fraichard, A., et al., J.Cell Science 108: 3161-3188, 1995), cardiomyocytes (heart muscle cells)(Klug, M., M. Soonpaa, and L. Field, Am. J. Physiol. 269: H1913-H1921,1995), skeletal muscle cells (Rohwedel, J., et al., Dev. Biol. 164:87-101, 1994), and vascular cells (Wang, R., R. Clark and V. Bautch,Development 114: 303-316, 1992). The factors responsible for maintainingthe pluripotency of ES and EGs remain poorly characterized and are oftendependent upon the species from which the cells have been harvested.

Subsequent to the work with mouse embryos, several groups have attemptedto develop stem cell lines from sheep, pig, and cow. A cell line withembryonic stem cell-like appearance has reportedly been cultured fromporcine embryos using culture conditions similar to mouse (M. Evans etal., PCT Application WO90/03432; E. Notarianni et al., J. Reprod. Fert.,Suppl. 41: 51, 1990; J. Piedrahita et al., Theriogenology 34: 879, 1990;E. Notarianni et al., Proceedings of the 4th World Congress on GeneticsApplied to Livestock Productions, 58, Edinburgh, July 1990). Othergroups have developed avian stem cell lines from chickens (Pain et al.,Dev. 122:1996).

To date, there have been no reports for the establishment of human EGcells or cell lines. Any method which would allow production of human ESand EG would be desirable since, human EG cell lines would permit easierstudy of early human development, and the use of such human EG celllines would enable the development of cell cultures for transplantation,manufacture of bio-pharmaceutical products, and development ofbiological-based sensors.

SUMMARY OF THE INVENTION

The present invention provides a human pluripotent embryonic germ cell(hEG) line and a method of producing cells exhibiting an hEG phenotype.hEGs are derived from EGs isolated from gonadal tissues, genital ridges,mesenteries or embryonic yolk sacs of human embryos and cultured underconditions which allow long term cell culture (more than 30 days). Theresulting hEG cells resemble ES cells in morphology, biochemicalhistotype and in pluripotency. These cells can be passaged andmaintained for several months in culture and can survivecryopreservation.

One object of the invention is to provide a method for producing humancell lines which exhibit an ES cell-like phenotype. Another object ofthe invention is to provide human pluripotential embryonic germ cell(hEG) lines in general, as well as differentiated cell lines derivedfrom hEGs. Another object is to provide human transgenic cells, celllines, or tissues using the hEGs of the invention. Another object is toprovide hEG cells or hEG derived stem cells of restricted developmentallineage for transplantation.

In one aspect, the invention provides a method for screening, compounds,including small molecules, that affect hEG cell function. The methodincludes incubating components comprising the compound and at least onehEG cell under conditions sufficient to allow the components tointeract; and determining the effect of the compound on an hEG cellfunction before and after incubating in the presence of the compound. Acell function that may be modulated (e.g., inhibited or stimulated) bythe compound includes differentiation, gene expression, production ofgrowth factors, response to growth factors and modulation of cellmembrane permeability. Yet another object is to provide usefulpharmaceutical products produced by the cells or cell lines of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows phase contrast microscopy photographs of human embryonicgerm cells (hEGs) showing positive histological staining for alkalinephosphatase.

FIG. 1a shows a non-motile PGC with characteristic rounded morphology;and

FIG. 1b shows a migratory PGC with characteristic psuedopodalmorphology.

FIG. 2 shows phase contrast microscopy photographs of human embryonicgerm cell colonies.

FIG. 2a demonstrates a morphology characteristic of a multilayer EGcolony.

FIG. 2b demonstrates a morphology characteristic of a monolayer EGcolony.

FIG. 3 shows phase contrast microscopy photographs of human embryonicgerm cell (hEG) colonies showing positive immunohistochemical stainingfor: (A) stage specific embryonic antigen-1 (SSEA-1); (B) stage specificembryonic antigen-3 (SSEA-3); (C) stage specific embryonic antigen-4(SSEA-4); (D) a cell surface antigen that binds with the antibody havingthe binding specificity of the monoclonal antibody designated TRA-1-60;(E) a cell surface antigen that binds with the antibody having thebinding specificity of the monoclonal antibody designated TRA-1-81; (F)alkaline phosphatase activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of a way of producinghuman EG cells in culture. An advantage of the invention is that largenumbers of hEG cells can be quickly and efficiently produced. In anillustrative example provided herein, the starting material isprimordial germ cells isolated over a period of 3-13 weeks postfertilization, from human fetal/embryonic yolk sac, mesenteries, andgonadal ridges, successively. Alternatively, gonocytes of latertesticular stages are utilized. The development of hEG cell culturesthat can be maintained as cell lines permits investigation offundamental questions regarding the biochemical and cellular propertiesof these cells and the dynamics of interaction in their cellular andchemical environment.

The hEG cells of the invention can advantageously be used to stablyincorporate genetic sequences encoding various receptors, ligands andneurotransmitters, for example, for use in the treatment of subjectswith various disorders and for identifying compounds and small moleculesthat interact with the genetically modified cells or the hEG cellsthemselves.

Before the present human cells expressing an embryonic pluripotentphenotype, compositions, reagents and methods and uses thereof aredescribed, it is to be understood that this invention is not limited tothe cells, compositions, reagents, methods or uses described, as suchcells, compositions, reagents, methods or uses may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and that theterminology used herein is not intended to limit the scope of thepresent invention which will only be limited by the appended claims.

The publications discussed above are provided solely for theirdisclosure before the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.Throughout this description, the preferred embodiment and examples shownshould be considered as exemplars, rather than as limitations on thepresent invention.

In one embodiment, the invention provides a method of producingpluripotent human cells exhibiting an embryonic cell phenotype. Thestarting material for isolating the cells may be primordial germ cellsisolated over a period of about 3-13 weeks post-fertilization, or morepreferably from about 5-9 weeks post-fertilization, from embryonic yolksac, mesenteries, or gonadal ridges, successively from humanembryos/fetus. In another embodiment, gonocytes of later testicularstages are isolated. The primordial germ cells (PGCs) are cultured onmitotically inactivated fibroblast cells (e.g., STO cells) underconditions in long term cell culture (more than 30 days) to allow theproduction of hEGs. The resulting cells resemble human ES cells inmorphology and in biochemical histotype. The cells can be passaged,maintained for several months in culture and survive cryopreservation.

Specifically, the present invention describes two human pluripotentialembryonic germ cell (hEG) cultures, designated hEG-KH and hEG-GU. ThehEG-KH and hEG-GU cell cultures are derived from the gonadal anlagen orgenital ridges of approximately 8 and 11-week last menstrual period(LMP) aborted human fetal material, respectively. Considering the sourceand colony morphology of these cells, they most resemble or are humanpluripotent embryonic germ cells (hEGs).

Human pluripotential stem cells (hEG) are human cells that can becultured indefinitely in an undifferentiated state, yet retain theability to be differentiated into a variety of cell and tissue types. Inaccordance with the present invention, the terms “pluripotent” and“pluripotential cells” refer to those cells which retain thedevelopmental potential to differentiate into a wide range of celllineages including the germ line. The terms “embryonic stem cellphenotype” and “embryonic stem-like cell” also are used interchangeablyherein to describe cells which are undifferentiated and thus arepluripotent cells and which are visually distinguished from other adultcells of the same animal.

The ability of hEG cells to differentiate in vitro into a wide varietyof cell types including the ability to differentiate into embryonic andmore highly differentiated cell types which can easily be tested bymeans common to those in the art. For example, to induce differentiationin monolayer cultures hEG cells are cultured for 2 weeks without passageonto a fresh fibroblast, e.g., STO, feeder layer. To inducedifferentiation in suspension culture, the cells are passed onto agelatinized plate to eliminate possible contamination by fibroblasts.After 4 to 7 days in culture, colonies are gently dislodged from theplate and disaggregated after incubation in 0.25% trypsin-EDTA for 10-15min. Dissociated cells are cultured in a microdrop of hEG culture mediumcontaining 0.3 μM retinoic acid on a 35-mm nonadhesive petridish.Suspension cultures are monitored daily for embryoid body formationwhich is indicative of a differentiated phenotype. (Similar experimentstesting for differentiation of attached hEG cells are well known tothose in the art.) Cell culture media is changed every other day. Basedon the resulting differentiated morphological types putative hEG cellscan be tested for their pluripotency.

The term “primordial germ cells” is used to describe undifferentiatedembryonic cells that will give rise to gametes. In another embodiment,gonocytes of later testicular stages are isolated. The resulting hEGcells resemble ES cells in morphology, biochemical histotype and inpluripotency. These cells can be passaged in culture, maintained forseveral months in culture, and survive cryopreservation. By the term“anlagen” is meant the rudiment or the primordia of an organ, tissue orpart thereof. The term “cell” as used herein also refers to individualcells, cell lines, or cultures derived from such cells. The term“embryonic germ cell” is used to describe cells of the present inventionthat exhibit an embryonic pluripotent cell phenotype. The term “cellline” as used herein refers to hEG cells or cells derived therefrom suchas are maintained in vitro culture. A cell line is substantially free ofother cells.

The terms “human embryonic germ cell (hEG)” or “embryonic germ cell” canbe used interchangeably herein to describe human cells, or cell linesthereof, of the present invention that exhibit a pluripotent embryonicstem cell phenotype that have particular characteristics. For example,in one embodiment, the hEG is pluripotent and is characterized by thepresence of markers associated with specific epitopic sites identifiedby the binding of particular antibodies and the absence of certainmarkers as identified by the lack of binding of certain antibodies. Inanother embodiment, the hEG is dependent on some growth factors formaintenance in the cultured state. Growth factors, as defined herein,are intercellular signaling polypeptides which control both thedevelopment and maintenance of cells, and the form and function oftissues. Preferably, the growth factor used in the method of the presentinvention is basic growth factor (bFGF). In another embodiment, the hEGrequires a ligand which binds to a receptor on hEG that canheterodimerize with glycoprotein 130 (gp130). The ligand is oncostatin-Mor leukemia inhibitory factor (LIF). In addition, an antibody that bindsto and activates gp130 directly can be utilized.

The hEG stains positively for the presence of alkaline phosphatase (AP),therefore, AP is one measurement that can be used to identify hEG cells.The hEG cells also expresses cell surface antigens SSEA-1 and SSEA-4 andexpress cell surface antigens that bind to antibodies having the bindingspecificity of monoclonal antibodies TRA-1-60 and TRA-1-81. By “bindingspecificity” is meant to include any antibody that correspond to themonoclonal antibody TRA-1-60 or TRA-1-81. One antibody corresponds toanother antibody if both antibodies recognize the same or overlappingantigen binding sites as demonstrated by, for example, a bindinginhibition assay commonly known to those in the art (Antibodies: ALaboratory Manual; Harlow & Lane, Cold Spring Harbor Laboratory, currentedition). hEGs of the invention can also express the cell surfaceantigen SSEA-3. Depending upon the culture conditions, the hEG candifferentiate into a variety of mature adult cell phenotypes that stainpositively for particular biochemical markers and do not stain for otherbiochemical markers. Differentiated hEGs also exhibit, in still anotherembodiment, mature morphological features that enable one skilled in theart to distinguish them from non-differentiated hEGs.

In another embodiment, the invention provides a method to produce human“transplants” using hEG cells of the present invention. The term“transplants” is used to describe cells (or parts thereof), cellproducts, tissue, or cell culture products derived from hEG cells thatare grafted into a human host. Specifically, a transplant is produced bymanipulating hEGs, which exhibit a pluripotent embryonic germ cellphenotype, in vitro to produce hEG derived stem cells of restricteddevelopmental lineage. By the term “restricted developmental lineage” ismeant that the prospective fate of the stem cells derived from the hEGcell is reduced to a smaller number of possible histotypes afterinduction of differentiation. Methods of inducing in vitrodifferentiation of hEG cells such as using retinoic acid or by theremoval of cell feeder layers or conditioned media are commonly known tothose in the art. The resulting stem cells of restricted developmentallineage can be further manipulated to include exogenous genetic materialknown as a transgene. Provided that the cell expressing an hEG phenotypeis genetically manipulated to include exogenous material, the resultingtransplant can include exogenous material within some, but not all ofits cells. Resulting transplant cell lines of restricted developmentallineage can be maintained or further manipulated as pure cell lines bytechniques common to those in the art.

The term “transgenic” is used to describe an animal or any part thereof,including, but not restricted, to cells, cultures or tissues whichincludes exogenous genetic material within its cells. Cells of theinvention can have DNA added to them and these cells can then be used ina manner similar to that for making a chimeric organism.

“Transgene” means any piece of DNA inserted by artifice into a cell thatbecomes part of the genome of the cell, cell line, tissue or organism(i.e., either stably integrated or as a stable extrachromosomal element)which develops from that cell. Such a transgene may include a gene whichis partly or entirely heterologous (i.e., foreign) to the cell ororganism to which the heterologous gene is introduced, or may representa gene homologous to an endogenous gene of the organism. Included withinthis definition is a transgene created by the providing of an RNAsequence that is transcribed into DNA and then incorporated into thegenome. The term “transgenic” as used herein additionally includes anyorganism or any part thereof, including, but not restricted, to cells,cell lines, cell cultures or tissues whose genome has been altered by invitro manipulation or by any transgenic technology to induce a specificgene knockout. The term “gene knockout” as used herein, refers to thetargeted disruption of a gene with either partial or complete loss offunction achieved by any transgenic technology familiar to those in theart. In one embodiment, transgenic cells having gene knockouts are thosein which the target gene has been rendered nonfunctional by an insertiontargeted to the gene to be rendered nonfunctional by homologousrecombination. As used herein, the term “transgenic” includes anytransgenic technology familiar to those in the art which can produce anorganism, cell, cell culture, cell line, tissue or embryo carrying anintroduced transgene or one in which an endogenous gene has beenrendered nonfunctional or “knocked out.”

“Transfected” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of any recombinant nucleic acid techniquesknown to those in the art, a heterologous nucleic acid molecule.“Heterologous nucleic acid” refers to a nucleic acid sequence thateither originates from another species or is modified from either itsoriginal form or the form primarily expressed in a cell.

The term “culture medium” means a suitable medium capable of supportinggrowth of hEG cells. Examples of suitable culture media useful inpracticing the present invention are a variety of hEG growth mediaprepared with a base of Dulbecco's minimal essential media (DMEM)supplemented with 15% fetal calf serum, 2 mM glutamine, 1 mM sodiumpyruvate, or glucose and phosphate free modified human tubal fluid media(HTF) supplemented with 15% fetal calf serum, 0.2 mM glutamine, 0.5 mMtaurine, and 0.01 mM each of the following amino acids; asparagine,glycine, glutamic acid, cysteine, lysine, proline, serine, histidine,and aspartic acid (McKiernan, S. M. Clayton, and B. Bavister, MolecularReproduction and Development 42: 188-199, 1995). An effective amount offactors are then added daily to either of these base solutions toprepare hEG growth media of the instant invention. The phrase “effectiveamount” as used herein is the amount of such described factor as topermit a beneficial effect on hEG growth and viability of hEG cellsusing judgment common to those in the art of cell culturing and by theteachings supplied herein.

One class of factors are ligands for receptors that activate the signaltransduction gp130, either by binding to a receptor that heterodimerizeswith gp130 or by binding directly to and activating gp130. For example,human recombinant leukemia inhibitory factor (LIF) at about 1000 U/ml to2000 U/ml or oncostatin-M at 10 U/ml, can be used (Koshimizu, U., etal., Development 122: 1235-1242, 1996).

A second class of factors are those which elevate intracellular cAMPlevels. For example, one or more of the following factors can be used atthe stated final concentration: forskolin at 10 μM, cholera toxin at 10μM, isobutylmethylxanthine (IBMX) at 0.1 mM, dibutyrladenosine cyclicmonophosphate (dbcAMP) at 1 mM (Dolci, S., M. Pesce, and M. De Felici,Molecular Reproduction and Development 35: 134-139, 1993; De Felici, M.,S. Dolci, and M. Pesce, Developmental Biology 157: 277-280, 1993;Halaban, R., et al., 1993).

A third class of factors are growth factors. In one particularembodiment the growth factor is basic fibroblast growth factor (bFGF),and more specifically, human recombinant basic fibroblast growth factor(bFGF) in the range of about 1-10 ng/ml. A fourth factor is growth mediaharvested from the culture of human embryonal carcinoma (EC) cells. In aparticular embodiment, for example, human NTERA-2 EC cells (ATCCaccession number CRL 1973) are grown to confluence in DMEM supplementedwith 10% fetal calf serum or mouse ES cells are grown to confluence inDMEM supplemented with 15% fetal calf serum, 2 mM glutamine, 1000 U/mlLIF. Growth media is harvested daily over several days, passed through a0.22 micron filter and frozen at −80° C. This EC or ES “conditioned”media is added to the hEG growth media in empirically determinedamounts, as judged by the effect on hEG growth and viability.

The term “STO cell” refers to embryonic fibroblast mouse cells such asare commercially available and include those deposited as ATCC CRL 1503,and ATCC 56-X. After the hEG cells of the invention are isolated, theycan be maintained by methods of growth and maintenance of cells known inthe art. It is understood that other fibroblast cells can be used in themethod of the invention, as long as they can function as feeder cellsfor the production of hEG cells of the invention.

In one aspect of the invention, the pluripotent hEG cell lines offer avaluable paradigm for the immunohistological investigation of earlyhuman development by using monoclonal antibodies specific for cellsurface glycolipids (Chiquoine, A. D., Anat. Rec., 118: 135-146, 1954;Evans, M. J., and M. H. Kaufman, Nature 292: 154-156) and glycoproteins(Hogan, B. L. M., U. S. Pat. No. 5,453,357) of the cells of the presentinvention. These reagents are developed by immunization of mice withmouse and human teratocarcinoma cell lines, as well as mouse embryos. Anumber of monoclonal antibodies which bind to embryonic cell surfaceepitopes have been produced in this manner, and are important in theelucidation of glycosylation pathways during development. Monoclonalantibodies which bind to cell surface glycolipids and glycoproteins havebeen used to study human germ cell tumors (Labosky, P. A., D. P. Barlow,and B. L. M. Hogan, Development 120: 3197-3204, 1994; Matsui, Y., D., etal., Nature 353: 750-751, 1991) and other cancers (Resnick, J. L., etal., Nature 359: 550-551, 1992; Thomson, J. A., et al., Proc. Natl. Aca.Sci. USA 92: 7844-7848, 1992).

To generate human specific embryonic cell surface antibodies, mice areimmunized weekly with about 10⁶ to 10⁷ hEG cells, and tail-bled weeklyto test for reactivity to hEG cells. After reactive sera is detected,hybridomas are produced as described (Andrews, P., et al., Hybridoma 3:347-361, 1984) or by standard methods. Resultant monoclonal antibodiesare screened against a panel of human cell lines including EG and EC, aswell as tissue sections from a variety of germ cell tumors and normalhuman tissues. Mouse ES, EG, and EC lines are also examined. Othermethods of making antibody fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (current edition), incorporated herein byreference). The term “antibody” as used in this invention includesintact molecules as well as fragments thereof, such as Fab, Fab′,F(ab′)₂, and Fv that can bind the epitopic determinant. As used in thisinvention, the term “epitope” means any antigenic determinant on anantigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

If needed, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See, e.g., Coligan, etal., Current Protocols in Immunology, Wiley Interscience, currentedition, incorporated by reference).

“Purified antibody” means an antibody that is at least 60%, by weight,free from proteins and naturally-occurring organic molecules with whichit is naturally associated. Preferably, the preparation is at least 75%,more preferably 90%, and most preferably at least 99%, by weight,antibody, e.g., an anti-SSEA-1 specific antibody. A purified antibodymay be obtained, for example, by affinity chromatography usingrecombinantly-produced protein or conserved motif peptides and standardtechniques. The invention can employ not only intact monoclonal orpolyclonal antibodies, but also an immunologically-active antibodyfragment, such as a Fab, Fab′ or (Fab′)₂ fragments, or a geneticallyengineered Fv fragment (Ladner et al., U.S. Pat. No. 4,946,788).

The methodology and cells of the present invention have a variety ofdifferent uses. The cells can be used to study human embryologicaldevelopment. For example, the cells of the invention which exhibitembryonic stem cell phenotype can be manipulated with detectably-labeledmarkers. The markers can then be inserted into blastocysts to observedistribution and cell lineages during development of the embryo.

“Detectably-labeled” refers to any means for marking and identifying thepresence of a cell or part thereof, i.e., an oligonucleotide probe orprimer, an antibody or fragment thereof, a protein or fragment thereof,a gene or fragment thereof, or a cDNA molecule. Methods fordetectably-labeling cells or molecules are well known in the art andinclude, without limitation, radioactive labeling (e.g., with an isotopesuch as ³²P or ³⁵S) and nonradioactive labeling (e.g., chemiluminescentlabeling, fluorescent labeling, enzymatic reaction products coded bygenes, i.e., CAT).

Some additional advantages of using the cells of the invention whichexhibit a pluripotent embryonic germ cell phenotype are as follows: Atransgene of interest is introduced into an hEG cell or hEG cell line ofthe present invention by electroporation, calcium phosphate,microinjection, lipofection, retro- or other viral or microbial vectoror other means and its integration and expression characterized invitro. The effect of the introduced gene on the transformed hEG cell isthen studied in vitro. The site in the hEG cell genome at which theintroduced gene integrates can then be manipulated, for gene targetingand gene replacement (Thomas, K. R. and Capecci, M. R. Cell 51: 503-512,1987).

Additionally, the hEG cells or cell lines of the invention are a sourceof RNA for the construction of early development and human pluripotentembryonic germ cell cDNA libraries. Gene expression during the earlystages of human development, and in cells which retain pluripotency, hastraditionally been difficult to study due to the scarcity of pertinentnucleic acid, molecules, cells and tissues. Using the techniques of thepresent invention, one of ordinary skill in the art can overcome thesedifficulties by generating stage specific human nucleic acid, molecules,cells, tissues and genetic material.

Pharmaceuticals, diagnostics, or antibodies, used in manufacturing orprocessing, are also produced using cells of the present invention.Exogenous foreign or homologous DNA is transferred to hEG cells byelectroporation, calcium phosphate, microinjection, lipofection, retro-or other viral or microbial vector or other means. The hEG cells arescreened for incorporation of this DNA, or are used in nuclear transfersystems. These proteins or other molecules are harvested from resultingcell cultures for further purification. For example, human bloodclotting factor IX may be produced for treatment of hemophilia.

Non-limiting examples of the following pharmaceutical, therapeutic,processing, manufacturing or compositional proteins that may be producedin this manner include: blood proteins (clotting factors VIII and IX,complement factors or components, hemoglobins or other blood proteinsand the like); hormones (insulin, growth hormone, thyroid hormone,gonadotrophins, PMSG, trophic hormones, prolactin, oxytocin, dopamine,catecholamines and the like); growth factors (EGF, PDGF, NGF, IGF andthe like); cytokines (interleukins, CSF, GMCSF, TNF, TGFα, TGFβ, and thelike); enzymes (tissue plasminogen activator, streptokinase, cholesterolbiosynthetic or degradative, digestive, steroidogenic, kinases,phosphodiesterases, methylases, de-methylases, dehydrogenases,cellulases, proteases, lipases, phospholipases, aromatase, cytochromesadenylate or guanylate cyclases and the like); hormone or otherreceptors (LDL, HDL, steroid, protein, peptide, lipid or prostaglandinand the like); binding proteins (steroid binding proteins, growthhormone or growth factor binding proteins and the like); immune systemproteins (antibodies, SLA or MHC gene products); antigens (bacterial,parasitic, viral, allergens, and the like); translation or transcriptionfactors, oncoproteins or proto-oncoproteins, milk proteins (caseins,lactalbumins, whey and the like); muscle proteins (myosin, tropomyosin,and the like).

The nucleotide sequence of the transgene may encode a precursor form ofthe protein ultimately harvested from the transgenic or transformedcells or cell cultures of the present invention. Preferably, expressionof the transgene is inducible. Alternatively, cells may be screened bytechniques well known to those of ordinary skill in the art to determinethe expression of the transgene by using it as a probe for testing mRNAfrom cell lines.

Production of differentiated cells for replacement, repair oraugmentation of damaged, nonfunctional, or impaired cells or tissues areanother use and embodiment provided by the present invention. Exogenousforeign or homologous DNA is transferred to hEG cells byelectroporation, calcium phosphate, microinjection, lipofection, retro-or other viral or microbial vector or other means. The hEG cells arescreened for incorporation for this DNA or used in nuclear transfersystems. These cells and/or tissues are harvested from cell cultures, orresulting cell lines for use in repairing or augmenting a defect. Forexample, cells, cell products, tissues or the products of cell culturesmay be used in treating subjects having Parkinson's disease or subjectswho have had a heart attack or spinal cord injury.

Cells, tissues or organs with exogenous major histocompatibility orother foreign or endogenous antigens and/or genes that will decreaserejection by the host organism of these transplanted materials areproduced by means of the present invention. For example, members of theFas ligand gene family can be utilized. Exogenous foreign or homologousDNA is transferred to hEG cell phenotype by electroporation, exposure tocalcium phosphate, microinjection, lipofection, retro- or other viral ormicrobial vector, or other means. The hEG cells are screened forincorporation of this DNA or expression of antigens, used in nucleartransfer systems, or grown in vitro culture. Molecules, proteins, cells,tissues, organs, fluids, or cell products are harvested from cells, celllines, cell cultures for xenotransplantation. In this manner, humanizedmolecules, proteins, cells, cell products, cell constituents, tissues,organs or fluids are possible.

In another embodiment, the invention provides methods to generate cellsand tissues from hEG lines for human transplantation. Towards that end,it may be necessary to eliminate or reduce cell-surface marker moleculeson donor transplantation cells or tissues that induce organ graftrejection. The present invention encompasses all such modifications thatreduce or eliminate organ graft rejection when employing cells, celllines (or any parts or derivatives thereof) from the present invention.These molecules, termed HLA antigens in humans, comprise MHC class I andII membrane glycoproteins. For non-hematopoietic cells and tissues,elimination or reduction of MHC class I molecules is accomplished bytargeted knockout of the human β₂-microglobulin gene, as has beenaccomplished with mouse ES cells (Zijlstra, M., et al, Nature 342:435-438, 1989). Non-hematopoietic cells do not normally produce MHCclass II molecules. For hematopoietic cells, the presence of MHC classII glycoproteins may be reduced or eliminated by targeted knockout ofthe HLA-DP, -DQ, and -DR loci, which are analogous to knockouts of the Eand A loci in mouse ES cells (Cosgrove, D., et al, Cell 66: 1051-1066,1991).

In another embodiment, the invention provides a method for identifying acompound which modulates an hEG function in some way (e.g., modulatesdifferentiation, cell proliferation, production of factors or otherproteins, gene expression). The method includes: a) incubatingcomponents comprising the compound and hEG cell(s) under conditionssufficient to allow the components to interact; and b) determining theeffect of the compound on the hEG cell(s) before and after incubating inthe presence of the compound. Compounds that affect hEG cell functioninclude peptides, peptidomimetics, polypeptides, chemical compounds andbiologic agents. Differentiation, gene expression, cell membranepermeability, proliferation and the like can be determined by methodscommonly used in the art. The term “modulation” refers to inhibition,augmentation, or stimulation of a particular hEG cell function.

Incubating includes conditions which allow contact between the testcompound and the hEG cell or cells. Contacting includes in vitro and invivo. For example, it may be desirable to test an array of compounds orsmall molecules on a single or few hEG cells on a “chip” or other solidsupport. For example, cardiomyocytes or neurons on chips would give areadout of the rate of contraction or number of firings, respectively,in response to a compound and for the detection of harmful or at leastbiologically active environmental agents (e.g., toxins, nerve gas orother weapons). The test compound may optionally be a combinatoriallibrary for screening a plurality of compounds. Compounds identified inthe method of the invention can be further evaluated, detected, cloned,sequenced, and the like, either in solution or after binding to a solidsupport, by any method usually applied to the detection of a specificDNA sequence such as PCR, oligomer restriction (Saiki, et al.,Bio/Technology, 3: 1008-1012, 1985), allele-specific oligonucleotide(ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. USA,80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, etal., Science, 241:1077, 1988), and the like. Molecular techniques forDNA analysis have been reviewed (Landegren, et al., Science,242:229-237, 1988).

EXAMPLES

All references cited herein are hereby incorporated by reference intheir entirety. The following examples are intended to illustrate butnot limit the invention. While they are typical of those that might beused, other procedures known to those skilled in the art mayalternatively be used.

Example 1 Collection and Preparation of Human Primordial Embryonic GermCells

Gonadal anlagen or genital ridges with mesenteries were dissected from8-11 week LMP (last menstrual period) human aborted fetal material. Thegenital ridges were placed into approximately 0.5 ml phosphate bufferedsaline solution or other isotonic buffer (PBS 0.21 g/L KH₂PO₄; 9 g/LNaCL; 0.726 g/L Na₂HPO₄7H₂O), and were cut into small (less than 1 mm³)chunks. The chunks were then further minced with a fine forceps. Thetissues were then transferred to a 15 ml polypropylene conical tube, andallowed to settle. A majority of the PBS was then carefully removed, and1 ml 0.05% trypsin-0.53 mM Sodium EDTA solution (BRL) was added to thetube. The tissue was then repeatedly pipetted through a 100 ul pipet tipto further disaggregate the cells. The tubes were subsequently stored onice for less than 1 hour.

The tissue and cell suspension was incubated at 37° C. for approximately5 min., then 15 ml EG growth media (D-MEM, 4500 mg/L D-glucose, 2200mg/L mM Sodium bicarbonate); 15% ES qualified fetal calf serum (BRL); 2mM glutamine (BRL); 1 mM Sodium Pyruvate (BRL); 1000-2000 U/ml humanrecombinant leukemia inhibitory factor (LIF, Genzyme); 1 ng/ml humanrecombinant basic fibroblast growth factor (bFGF, Genzyme); 10 uMForskolin in 10% DMSO was added. The tissue and cell suspension was thenspun at 1000 rpm for 5 min. The EG growth media was carefully removed,and the cells were resuspended in 3.2 ml EG growth media.

Approximately 0.2 ml of the cell suspension was added to each of 16wells of a 96-well tissue culture plate previously prepared with a subconfluent layer of STO mouse fibroblasts that had been cultured for 3days in hEG growth media that did not contain LIF, bFGF or Forskolin,then irradiated with 5000 rad of gamma irradiation.

The human PGC cells and STO mouse fibroblasts were cultured for 7-10days in hEG growth media at 37° C. with 5% CO₂ at 90% humidity. Growthmedia was freshly prepared and replaced daily. Alternatively,subconfluent fibroblast cells can be irradiated, then plated into tissueculture plates to form a feeder layer.

The cells were trypsinized as described here, and each well was passagedto 1 well of a 24-well culture dish previously prepared with irradiatedSTO mouse fibroblasts (90% of the cells) and to 1 well of a 96-welltissue culture plate previously prepared with irradiated STO mousefibroblasts (10% of the cells).

The cells were cultured for 3 days with daily replacement of growthmedia. On the 13th day of culture (3 days after subculture), a subset ofcells growing on the 96-well culture dish were fixed and stained for thepresence of alkaline phosphatase by using a commercially availablediagnostic kit (Sigma Chemicals, product number 86-R). The cells arewashed 2 times with phosphate buffered saline (PBS) then fixed for 30seconds in Citrate-Acetone-Formaldehyde solution. Fixed cells are thenincubated in the dark for 15 min. in Alkaline-dye mixture. The cells arethen rinsed with deionized water for 2 min. and allowed to dry. Alkalinephosphatase positive PGC and EG cells stain red, while cells that lackalkaline phosphatase activity, such as STO cells, remain clear.

Cells growing on the 24-well plate were passaged four times to expandcell numbers, and multiple frozen stocks from each passage wereprepared. Cells were photographed throughout the initial 13 days ofculture using phase contrast microscopy and selected cells wereprocessed for alkaline phosphatase staining as described herein.

Example 2 Passage of Pluripotent Cells

hEG growth media was replaced daily, and the cells were grown at 37° C.,90% relative humidity, 5% CO₂ for 10-14 days. At this time, the cellswere trypsinized and subcultured to freshly prepared 96-well or 24-wellplates with irradiated feeder layer or matrix. A subpopulation of thesecells were fixed and stained for alkaline phosphatase activity. Thesecells were passaged at least 4 times over a 40 day period, withcontinued demonstration of alkaline phosphatase activity as demonstratedby positive staining.

After 1 to 3 passages (7 to 30 days) some cells in the human PGC culturechange from isolated and solitary human PGCs, readily identified only byalkaline phosphatase staining or antibody detection, to largemulticellular and compact clusters. These clusters can be recognized bylight microscopy and resemble early passage mouse ES and EG cells. Likethe solitary human PGC cultures, the multicellular cluster human EGcells can be characterized with respect to alkaline phosphataseactivity, presence of cell surface antigens, and ability to formteratocarcinomas in SCID mice. The multicellular clusters are isolatedfrom the rest of the culture using a cloning cylinder, expanded throughrepeated passage, then characterized as separate cell lines.

To test for tetracarcinoma formation in SCID mice, a pellet consistingof approximately 500,000 hEG cells is injected into the rear leg muscle,testis, or kidney capsule of 8-15 week old SCID mice. After 8-20 weeksof development, the resulting tumors are fixed in 4% paraformaldehydeand embedded in paraffin. Tissue sections are examined using standardhistological staining techniques and immunohistochemical detection ofcell surface antigens and other epitopes as described herein.

Example 3 Testing Harvested Cells for Morphology and Alkaline PhosphateActivity

As previously described, in a preferred embodiment primordial germ cells(PGCs) are harvested from nascent gonadal ridges since their earlydevelopmental age inhibits subsequent differentiation and loss ofpluripotency.

To ascertain that harvested cells are of an appropriate developmentalage, harvested cells were tested for morphological criteria used toidentify primordial germ cells that are pluripotent (DeFelici andMcLaren, Exp. Cell. 142: 476-482, 1982). To further substantiatepluripotency a sample of the extracted cells are subsequently tested foralkaline phosphatase (AP) activity. Markers for pluripotent cells areoften useful to identify stem cells in culture. hEG cells typicallymanifest alkaline phosphatase (AP) activity and AP positive cells aretypically pluripotent. AP activity is rapidly lost with differentiationof hEG cells in vitro. AP expression has been demonstrated in ES andES-like cells in the mouse (Wobus et al., Exp. Cell 152: 212-219, 1984;Pease et al., Dev. Bio. 141: 344-352, 1990), rat (Ouhibi et al., Mol.Repro. Dev. 40: 311-324, 1995), pig (Talbot et al., Mol. Repro. Dev. 36:139-147, 1993b) and cow (Talbot et al., Mol. Repro. Dev. 42: 35-52,1995). AP activity has also been detected in murine PGCs (Chiquoine,Anat. Rec. 118: 135-146, 1954), murine EG cells (Matsui et al, Cell 70:841-847, 1992; Resnick et al., Nature 359: 550-551, 1992) and culturedavian embryonic cells from chickens (Pain et al., Dev. 122:1996). Inconjunction with morphological evaluation of the hEG cell colony, APexpression is a convenient marker to identify pluripotent embryonic germcells in culture.

Cell samples taken from hEG cell lines cultured from 8-11 week LMPgonadal ridges (generated as in Example 2) were assessed, using lightmicroscopy, for the presence of morphological criteria indicative ofputative hEGs. The hEGs first appeared either as round cells or roundcells with two or more extended pseudopodia when visualized afterstaining for alkaline phosphatase (AP) activity After 1-4 weeks inculture, multicellular colonies of hEGs developed. First, 10 to 100individual cells formed a loosely associated aggregation. In subsequentpassages, some colonies became larger and appeared to be comprised ofmany cell layers. The individual cells of these colonies appeared smalland more tightly associated. hEG cultures are maintained for greaterthan 3 months by passage every 7-10 days onto fresh irradiated mouse STOfibroblasts. After approximately 2 months, some hEGs formed colonieswith larger diameter but fewer cell layers. The cells demonstrated atightly clustered and rapidly growing morphology reminiscent of earlypassage mouse ES and EG cultures.

Subsequently, cells were tested for alkaline phosphatase activity byfixing them in 80% ethanol (Buehr and McLaren, Meth. Enzymol. 225:58-77, 1993) and staining them employing a protocol from an APcytochemistry kit (Sigma Chemical Co., St. Louis, Mo.). The resultsindicated that AP activity was consistently expressed in hEGs, and inprimary cultures and subcultures of hEG cells. The cells demonstratedstrong and convincing histological staining for alkaline phosphatase.Surrounding STO mouse fibroblasts did not stain for alkalinephosphatase. Therefore, cells testing positive for both morphologicalcriteria and AP activity are indicative of ES-like cells and these cellstypically make up 50-90% of all harvested cells.

Example 4 Pluripotency of hEG Determined by Antibody Staining

Other indicators of pluripotency can also be investigated. Theseinclude, but are not limited to, the presence of stage specificembryonic antigens such as SSEA-1 (Solter, D. and B. Knowles, Proc.Natl. Acad. Sci. USA 75: 5565-5569, 1978), SSEA-3, SSEA-4 (Kannagi, R.,et al., Embo J. 2: 2355-2361, 1983) and epitopes recognized by theantibodies TRA-1-60 and TRA-1-81 (Andrews, P., et al., Hybridoma 3:347-361, 1984) and the ability of these cells to form teratocarcinomasor teratomas when injected into immuno-compromised (SCID) mice.

hEG cultures grown on plastic chamber slides were stained with 5monoclonal antibodies to embryonic cell surface antigens. Cells wererinsed 2 times with phosphate buffered saline (PBS) then fixed in 3%paraformaldehyde for 15 minutes at room temperature. The cells werewashed two times in PBS, then incubated in a 1:5 to 1:25 dilution (inPBS) of each of the following monoclonal antibodies for 1 hour at roomtemperature: TRA-1-81 and TRA-1-60 (supplied by Dr. Peter Andrews,Sheffield, UK); MC-480 (anti-SSEA-1), MC-631 (anti-SSEA-3), andMC-813-70 (anti-SSEA-4) (anti-SSEA antibodies were supplied by theDevelopmental Studies Hybridoma Bank, Iowa City, Iowa). The cells weresubsequently washed times two in PBS. Biotinylated anti-mouseimmunoglobulin secondary antibody, and horseradish peroxidase-conjugatedstrepavidin (BioGenex, San Ramon, Calif.) were used as recommended bythe manufacturer.

Antibodies to SSEA-1 and -4 antigens and TRA-1-60, and -81 reactedstrongly to the hEG cells, while the antibody to SSEA-3 reacted weakly.hEG cells also reacted positively for AP.

Example 5 Cultured hEG Cells Display Normal Karyotypes

Due to their rapid proliferation in culture established ES cells havebeen reported to contain abnormal karyotypes (Abbondanzo, S. J. et al.,Meth. Enzymol. 225: 803-823, 1993). Additionally, repeated freezing andthawing of cyropreserved ES cells may elevate the risk of inducingchromosomal abnormalities. To maximize the potential of successfulgerm-line genetic manipulation (e.g., gene targeting) when using hEGcells, hEG cell lines exhibiting normal diploid karyotypes arepreferred. To determine whether human hEG cell lines exhibited normalkaryotype, hEG cells which were cultured as described herein weretested. Approximately 10-20 metaphase stage karyotypes from each hEGcell line were tested by examining the cell's chromosomes for bothstructural and numerical abnormalities. Two hEG cultures were karyotypedand were normal 46,XY and 46,XX, respectively. Colonies from thesecultures were isolated and expanded two separate times to generate hEGlines.

Cells were placed in 4-well culture dishes and cultured overnight in hEGculture medium containing 0.02 ug/ml colcemid (GIBCO BRL) at 39° C. in5% CO₂, 95% air. Cells were subsequently washed in PBS, treated with0.25% trypsin-EDTA for 10-15 minutes at 39° C., removed and centrifugedfor five minutes at 800×gravity. Cells were fixed for five minutes incold Carnoy's fixative (3:1 volume of absolute methanol to glacialacetic acid), washed in PBS, centrifuged as above, and resuspended in0.5 ml of Carnoy's fixative. A pipette drop of the resulting cellsuspension was transferred onto microscopic slides that were prewashedwith Carnoy's fixative. Slides were air dried, Giemsa stained (GIBCO,BRL) and rinsed with tap water. After a second drying, slides were coverslipped and viewed under oil immersion using light microscopy at 400×magnification.

All hEG cell lines examined had a normal complement of human chromosomes(i.e., 44 autosomes and 2 sex chromosomes). Additionally, no breaks,deletions, additions or other abnormalities in the shape or number ofchromosomes were observed. Additionally, hEG cells that survivedcryopreservation and subsequent culturing also displayed no chromosomalabnormalities or overt changes in phenotypic characteristics. Amongisolated hEG cell lines, no obvious differences were observed inmorphology, proliferation and AP activity. The hEG cells expressed APactivity, as consistently observed in EGs and embryonic germ cells inprimary culture and subcultures but not in STO feeder cells. When thehEG cells differentiated in vitro, they rapidly lost AP activity. After8 to 12 passages, all 4 isolated hEG cell lines had a normal humancomplement of 46 chromosomes (44 autosomes and 2 sex chromosomes). Noobvious abnormalities, additions or deletions are found in chromosomesfrom isolated hEG cells as described above.

TABLE 1 Characteristics of human EG cell lines No. of hEG Cell KaryotypeCurrent Line Collected From (2N) Passage KH 9 week LMP 46,XX 20 GU 11week LMP 46,XY 30

Example 6 Use of Human Pluripotent Embryonic Germ Cells (hEG) toGenerate a cDNA Library

hEG cells of the present invention are a plentiful source of pluripotentcell mRNA to create cDNA libraries and templates for polymerase chainreaction based experimentation. hEG lines were cultured in the presenceof irradiated mouse STO fibroblasts. Several steps were taken toeliminate STO cells and STO cDNA. Approximately 10⁶ hEG cells growing onirradiated STO fibroblasts were trypsinized and resuspended in hEGmedia. The resuspended cells were plated on a tissue culture dish andallowed to sit for 1 hour. During this time, STO fibroblasts adherewhile hEG cells do not. Unattached cells were gently removed and theplating procedure was repeated twice. This series of preferentialbindings effectively removes 50-90% of STO cells. Remaining hEG cellswere spun at 1000 rpm for 5 minutes, and the pellet was used to generateRNA, mRNA and then cDNA. The cDNA was subjected to several rounds ofsubtraction using STO cell RNA, by a commonly described methodology(Maniatis, et al., Molecular Cloning. A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. current edition,incorporated herein by reference in its entirety). This removed STO andfibroblast cDNAs. The remaining cDNA was enriched for the human cDNAsunique to pluripotent cells. Many cDNA library screenings can beemployed on this cDNA library, as well as other DNA subtractionscommonly known to those in the art (Maniatis, et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., current edition, incorporated herein by reference, in itsentirety).

Example 7 Culture of hEG Cells

In the mouse, pluripotent embryonic stem cells are derived principallyfrom two sources. Embryonic stem (ES) cells are derived from the innercell mass of pre-implantation embryos, while embryonic germ (EG) cellsare derived from primordial germ cells (PGCs) located in the genitalridge of day 8.5 to 12.5 post coitum embryo. Both types of cells arepluripotent and demonstrate germline genetic transmission in the mouse.Mouse ES and EG cells share several morphological characteristics suchas high levels of intercellular alkaline phosphatase (AP), growth astightly associated multicellular colonies, presentation of specific cellsurface glycolipid and glycoprotein molecules. Some additionalcharacteristics are a normal and stable karyotype and the ability to becontinuously passaged. Embryonic stem cells that share some of thesecharacteristics have been derived from avian species, mink, hamster,pig, bovine and the rhesus monkey.

When allowed to differentiate, mouse ES and EG cells will differentiatein vitro and in vivo. With the proper combinations of growth anddifferentiation factors, they can generate cells of the hematopoieticlineage and caridomyocytes. Additionally, mouse ES cells have been usedto generate in vitro cultures of neurons, skeletal muscle, and vascularendothelial cells. When undifferentiated ES and EG cells are injectedinto mice (immunocompromised if appropriate), a teratocarcinoma forms atthe site. These tumors contain undifferentiated cells and a wide varietyof differentiated cell types.

Human pluripotent stem cell cultures and their lineage restrictedderivatives could potentially be used as an unlimited source of cellsand tissues for transplantation, bio-manufacturing, and developmentalresearch. Genetic manipulation of these cells may provide suitablevectors for future gene therapy approaches. In an effort to generatehuman pluripotent stem cells, we have initiated and characterized anumber of cell cultures derived from human PGCs.

Methods

Gonadal ridge and mesenteries of 5-9 week post-fertilization humanembryos were dissociated with 0.25% trypsin-EDTA and mechanicaldisruption. Tissues were initially cultured, and subsequently passaged,on an irradiated mouse STO fibroblast feeder layer in DMEM supplementedwith 15% FBS, human recombinant leukemia inhibitory factor (hrLIF),human recombinant basic fibroblast growth factor (hrbFGF) and forskolin.For alkaline phosphatase activity detection, cells were fixed in 66%acetone/3% formaldehyde then stained with napthol/FRV-alkaline APsubstrate (Sigma). For immunocytochemistry, cells were fixed in 3%buffered paraformaldehyde. Antibody detection was done usingbiotinylated anti-mouse antibodies, strepavidin conjugated horseradishperoxidase, and AEC chromagen (BioGenex). Cells prepared for cytogeneticanalysis were treated with 0.1 ug/ml Colecimd, 0.075M KCl, then 3:1methanol acetic acid fix.

Results

Pluripotent embryonic stem cell lines have been derived from cultures ofmouse primordial germ cells (PGCs), and have been referred to as EG(embryonic germ) cells. With the goal of establishing human EG celllines, gonadal ridge and mesenteries of 5-9 week postfertilizationembryos (obtained as the result of pregnancy termination) were culturedon mouse STO fibroblast feeder layers in the presence of a variety ofgrowth factors, including human recombinant leukemia inhibitory factor(hrLIF), human recombinant basic fibroblast growth factor (hrbFGF), andforskolin. Initially, single PGCs were visualized by alkalinephosphatase (AP) staining. Over a period of 7-21 days, these PGCs gaverise to large multicellular colonies resembling those of early passagemouse EG and embryonic stem (ES) cell colonies. Throughout the cultureperiod and with subsequent passages, the cells continued to be APpositive. The cells were also positive when tested against a panel offive monoclonal antibodies (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81)used routinely to characterize pluripotent stem cells. The culturedcells have been continuously passaged and found to be karyotypicallynormal and stable. Both XX and XY cell cultures have been obtained. Theproperties so far characterized on the derived human cells areconsistent with those anticipated for pluripotent stem cells. (See Table2)

Several human PGC derived cell cultures have been obtained. All culturestested have shared the morphological, immunological, and karyotypiccharacteristics described. In order to compare these cell cultures to ESor EG cells, their potential to differentiate in vitro and in vivo mustbe determined. During standard culture, a small fraction of coloniesspontaneously differentiate into structures that strongly resemble mouseembryoid bodies (EB). When analyzed by electron microscopy, a widevariety of cell types were identified, including an epithelial outerlayer covering an partially solid core of fibroblasts, endothelialcells, and what appear to be anucleated red blood cells.

TABLE 2 Reactivity Antibody Primate Mouse Name Antigen Antigen typehuman PGC derived hEC mES EC ES EC MC480 SSEA-1 glycolipid (lacto) + −− + + + MC631 SSEA-3 glycolipid (globo) +/− + + − − − MC813-70 SSEA-4glycolipid (globo) + + + − +/− + TRA-1-60 glycoprotein + + + − − −TRA-1-81 glycoprotein + + + − − −

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. An in vivo method for determining the effect of acompound on a human pluripotent embryonic germ (hEG) cell functioncomprising: a) incubating the compound and at least one hEG cell underconditions sufficient to allow the compound and the cell to interact,wherein the hEG cell is dependent on a growth factor and a factor whichbinds to a receptor which can heterodimerize with gp 130 duringmaintenance; and b) determining whether the compound has an effect oncell function compared to hEG cells not treated with the compound. 2.The method of claim 1, wherein the effect is inhibition of a cellfunction.
 3. The method of claim 1, wherein the effect is stimulation ofa cell function.
 4. The method of claim 1, wherein the compound is apeptide.
 5. The method of claim 1, wherein the compound is apeptidomimetic.
 6. The method of claim 1, wherein the at least one hEGcell is on a solid support.
 7. The method of claim 6, wherein the solidsupport is a chip.
 8. The method of claim 1, wherein the hEG cellfunction is selected from the group consisting of differentiation, cellproliferation, production of a protein, gene expression, production of agrowth factor, response to a growth factor and modulation of cellmembrane permeability.
 9. The method of claim 1, wherein the hEG cell isan isolated hEG cell.
 10. The method of claim 9, wherein the hEG cell isSSEA-1 positive.
 11. The method of claim 1, wherein the hEG cell isSSEA-1 positive.
 12. The method of claim 1, wherein the hEG cellfunction comprises differentiation into a restricted developmentallineage cell.
 13. The method of claim 12, wherein the restricteddevelopmental lineage cell is selected from the group consisting of aneuron, a vascular endothelial cell, a cardiomyocyte, a hematopoieticcell, and a skeletal muscle cell.
 14. The method of claim 1, wherein thehEG cell is dependent on a factor that elevates cAMP.
 15. The method ofclaim 14, wherein the factor that elevates cAMP is selected from thegroup consisting of forskolin, cholera toxin, isobutylmethylxanthine anddibutyladenosine cyclic monophosphate.
 16. The method of claim 15,wherein the factor that elevates cAMP is forskolin.